Photoinduced fluorescence switching of diarylethene derivatives at the single-molecule levelMiyasaka Lab.ARAI Yuhei11Contents.IntroductionSingle-Molecule Measurements (SMM) Microscope

. Applications of single molecule fluorescence imaging. My workMotivationMethodResult and Discussion

. Summary

2Wide-field

2Single-Molecule Measurements(SMM)3Each guest molecule is in different environment.

SpectrumSpectrum of ensembleSpectrum of a single molecule

TrajectoryTrajectory of a single molecule3Wide-fieldConfocalTime resolution30 fpm(16.7m16.7m)200 s(15 m15 m)Spatial resolution(x-y plane)250 nm250 nmSpatial resolution(z axis)3 m900 nmAdvantageMeasure many molecules at one timeHigh spatial resolution about z axisDisadvantageBackground light from out of focusLong measurement timeMeasure few molecules at one timeMicroscope4

Confocal microscopeWide-field microscopeZZ4Applications of SMM ~ /2sinDiffraction-limitConventional optical microscope Spatial Resolution is limited by diffraction-limit ~ about a half of wavelength ( > 200nm)

Super resolution microscopy Beyond the diffraction-limit~ from several to tens of nm

PhotoActivated Localization Microscopy (PALM)Using localization method and photo switchable fluorescent molecule

Stochastic Optical Reconstruction Microscopy (STORM)

diffraction-limit : 200nmnmnmPALM

5Applications of SMM

Stefan W. Hell, et al, Science,316 (2007) 1153.Fluorescent ON state partlyhv (activation)LocalizationHigh spatial resolved image(several nmten-odd nm)hv (excitation)OFF stateLocalizationSuper resolution microscopy(PALMPhoto-Activated Localization Microscopy)PALMnm

6Applications of SMM

X347.7780.06 pix.Y : 301.8470.06 pix.

Single-Molecule TrackingSMTsX, sY : Width of Gauss functionRotation anglebgbackground noiseI0Fluorescence intensityX0, y0 : Center of Gauss function

SMTSMT2

7Contents.IntroductionSingle-Molecule Measurements (SMM) Microscope

. Applications of single molecule fluorescence imaging. My workMotivationMethodResult and Discussion

. Summary

8SMT8MotivationEvaluating microscopic inhomogeneity of polymer film by using Single-molecule trackingPolymer chain is densePolymer chain is sparseMicroscopic structure of polymerLithographic nanofabricationDiffusional motion is fastDiffusional motion is slow

Nano imprinting

S.Takei et al, JJAP, 46(2007) 7279-7284http://www.suss.com/SMT9

Sample10Vis. (co